Evaluation of antimicrobial and antibiotic resistance properties of microbial community in a traditional cheese

The presence of antibiotic-resistant genes in local cheeses is not desirable due to the risk of gene migration, increased resistance to antibiotics in consumers, and therefore the effectiveness of related drugs during the treatment period. Recently, the studies showed that the demand of local cheeses made from raw milk have increased. However, there is concern that consumption of these cheeses could increase the risk of antibiotic-resistant genes transfer through bacteria especially Enterococcus spp to consumers. Of course, traditional fermented products may contain intrinsic microbial flora with unique useful properties, including the production of antimicrobial metabolites and resistance to phages, which is very important for the dairy industry. The ability of bacteriocin production by the microbial flora that responsible for fermentation of cheeses made from raw milk is very important because it prevents the growth of pathogenic bacteria. Therefore, the aims of this study were the evaluation of the presence of antibiotic-resistant microbial flora of local cheeses made from raw milk and the investigation of the useful properties such as the presence of its antimicrobial metabolites.

Cheese made from raw milk was produced based on a traditional recipe. For isolation of Enterococcus spp., KAA agar was used, followed by incubation for 48 h at 37°C. The serial dilution of cheese was carried out until the final dilution of 10-8 in ringer. For isolation of Enterococcus spp., a 100 µl of diluted sample was cultured on KAA agar, then incubated for 48 h under anaerobic conditions at 37°C. The catalase-negative and Gram-positive isolates were phenotypically distinguished at genus level using physiological tests )growth at salt concentrations 6.5% and 18%, pH 9.6 and 4.4, temperatures 10°C and 45°C and gas production. After phenotypic detection of isolates at genus level, culture-dependent characterization through PCR and subsequently sequencing was carried out. DNA extracted from cheese was proliferated and sequenced by NGS technique. The metagenomics data were processed for phage resistance, antibiotic resistance, bacteriocin and antioxidant components production. Furthermore, antibiotic resistance of isolates and antimicrobial properties of cell free supernatant (CFS) from cheese were evaluated.

A total of 20 bacteria were isolated from cheese and molecularly identified as Enterococcus malodoratous (60%), Enterococcus faecalis (5%), Enterococcus duran (5%), Enterococcus spp. (30%). Also, the metagenomic analysis showed that Enterococcal community of cheese include Enterococcus malodoratous (72%), Enterococcus faecalis (6%), Enterococcus italicus (13%), Enterococcus spp. (10%) and its microbiome is resistance to ‌-antibiotics ‌and bacteriophages and has the production potential of antioxidant compounds and antimicrobial compounds such as plantaricin. Moreover, laboratory tests confirmed ‌‌antibiotics resistance and antimicrobial properties of isolates.

This study showed that some local cheeses can be responsible for transferring antibiotic-resistant genes to humans and consumption of these cheeses should be limited.